Nuclear Localization Signal and p53 Binding Site in MAP/ERK Kinase Kinase 1 (MEKK1).
Nuclear Localization Signal and p53 Binding Site in MAP/ERK Kinase Kinase 1 (MEKK1).
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DOI：
10.1002/jcb.25238
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发表时间：
2015-12
影响因子：
4
通讯作者：
Islam MR
中科院分区：
文献类型：
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作者：
Chipps E;Protzman A;Muhi MZ;Ando S;Calvet JP;Islam MR
之前，我们发现 Mekk1 易位到细胞核，与肿瘤抑制蛋白 p53 相互作用，并通过最小 PKD1 启动子上的非典型 p53 结合位点共同抑制 PKD1 转录 (JBC 285:38818-38831, 2010)。在这项研究中，我们报告了 Mekk1 核转运和 p53 结合的机制。使用 GFP 连接的组成型活性 Mekk1 (CA-Mekk1) 和删除策略，我们鉴定了位于 C 端 Mekk1 催化结构域中氨基酸 (aa) 残基 1349-1353 处的核定位信号 (HRDVK)。 CA-Mekk1 和全长 Mekk1 中该序列的删除显着减少了 HEK293T 和 COS-1 细胞中的核转位。使用免疫共沉淀，我们鉴定了 Mekk1 中负责 p53 结合的相邻序列（GANLID，aa 1354-1360）。删除该序列显着减少了 Mekk1 与 p53 的相互作用。 Mekk1 似乎不会影响位于 Mdm2 相互作用位点的 Ser15 或 p53 中其他 Ser 残基的磷酸化。然而，Mekk1 在 Mdm2 存在的情况下介导 p53 蛋白稳定性并减少 p53 泛素化，表明通过泛素-蛋白酶体途径干扰 Mdm2 介导的 p53 降解。
Previously, we showed that Mekk1 translocates to the nucleus, interacts with tumor suppressor protein p53 and co-represses PKD1 transcription via an atypical p53 binding site on the minimal PKD1 promoter (JBC 285:38818-38831, 2010). In this study, we report the mechanisms of Mekk1 nuclear transport and p53 binding. Using GFP-linked constitutively active-Mekk1 (CA-Mekk1) and a deletion strategy, we identified a nuclear localization signal (HRDVK) located at amino acid (aa) residues 1349–1353 in the C-terminal Mekk1 catalytic domain. Deletion of this sequence in CA-Mekk1 and full-length Mekk1 significantly reduced their nuclear translocation in both HEK293T and COS-1 cells. Using co-immunoprecipitation we identified an adjacent sequence (GANLID, aa 1354–1360) in Mekk1 responsible for p53 binding. Deletion of this sequence markedly reduced the interaction of Mekk1 with p53. Mekk1 does not appear to affect phosphorylation of Ser15, located in the Mdm2 interaction site, or other Ser residues in p53. However, Mekk1 mediates p53 protein stability in the presence of Mdm2 and reduces p53 ubiquitination, suggesting an interference with Mdm2-mediated degradation of p53 by the ubiquitin-proteasome pathway.